Methods for prenatal diagnosis of chromosomal abnormalities

Abstract

Chromosomal abnormalities are responsible for a significant number of birth defects, including mental retardation. The present invention is related to methods for non-invasive and rapid, prenatal diagnosis of chromosomal abnormalities based on analysis of a maternal blood sample. The invention exploits the differences in DNA between the mother and fetus, for instance differences in their methylation states, as a means to enrich for fetal DNA in maternal plasma sample. The methods described herein can be used to detect chromosomal DNA deletions and duplications. In a preferred embodiment, the methods are used to diagnose chromosomal aneuploidy and related disorders, such as Down's and Turner's Syndrome.

1 . A kit for detecting chromosomal aneuploidy in the maternal plasma sample, the kit comprising one or more enzymes to specifically digest the maternal DNA in the plasma sample of a pregnant female, and primers to detect paternal and maternal allele frequency of polymorphic markers in the enriched fetal DNA regions for detection of chromosomal deletions, insertions or aneuploidy. 
     
     
         2 . The kit of  claim 1 , comprising two or more enzymes. 
     
     
         3 . The kit of  claim 1  or  2 , wherein the enzymes are methylation sensitive enzymes. 
     
     
         4 . The kit of  claim 1  further comprising at least one control DNA isolated from plasma of a mother pregnant with a healthy fetus, and/or DNA samples isolated from plasma samples from females carrying a fetus with a chromosomal abnormality. 
     
     
         5 . The kit of  claim 4 , wherein the chromosomal abnormality is chromosome 21, 13, or 18 trisomy. 
     
     
         6 . A kit for prenatal diagnosis of chromosomal abnormalities, the kit comprising at least one methylation-sensitive enzyme, at least one pair of DNA amplification primers capable of annealing and thus amplifying regions flanking sites that contain at least one polymorphic locus within differentially methylated regions in fetal and maternal DNA present in maternal plasma, at least one primer or probe to allow detection of alleles in the at least one polymorphic locus, and an instruction manual instructing the user to perform the steps of selectively digesting the DNA present in a plasma sample from a pregnant woman with the methylation-sensitive enzyme to enrich the fetal DNA in the sample, performing DNA amplification using the amplification primers and detecting the alleles present in the sample enriched for the fetal DNA, and interpreting the results so that if the ratio of two different alleles in the locus deviates from a control wherein the alleles are present in equal amounts, the fetus is affected with a chromosomal abnormality. 
     
     
         7 . The kit of  claim 6 , comprising primers to amplify 5-10 or more polymorphic markers for an analysis when parental alleles are not known and allowing identification of at least one informative marker to perform the diagnosis of chromosomal abnormality. 
     
     
         8 . The kit of  claim 7 , wherein the polymorphic markers are selected from chromosomes 21, 13, and 18. 
     
     
         9 . The kit of  claim 6 , further comprising a control nucleic acid panel comprising nucleic acids isolated from females pregnant with fetuses carrying known chromosomal abnormalities and females pregnant with fetuses without chromosomal abnormalities. 
     
     
         10 . The kit of  claim 7  further comprising an internal control of at least one pair of amplification primers and a detection primer or probe, wherein the primers and/or probe are selected from a nucleic acid region that is differentially methylated in fetal and maternal DNA present in maternal plasma, but that occur in chromosomes, wherein duplication or deletion is rare, so as to provide an internal control.